ABSTRACT. Reactivation of latent human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality in transplant recipients, despite the use of antiviral drugs. Therefore, new approaches are required to reduce the complications from this pathogen. Previous studies have shown that HCMV establishes latency in myeloid lineage cells, including CD34+ hematopoietic progenitor cells (HPCs). These pluripotent cells have a unique epigenetic environment in which HDAC expression is low, and many genes are transcriptionally inactive, but carry bivalent chromatin marks (repressive H3K27me3 and activating H3K4me3) characteristic of facultative chromatin. We hypothesize that HCMV exploits the unique epigenetic environment of HSCs, so that most lytic genes are repressed through bivalent histone modification, but the viral genome is poised to reactivate under appropriate stimulation. The major immediate early (IE) genes encode transcriptional regulatory proteins required to activate lytic infection. Expression of these genes is controlled by the major immediate early promoter (MIEP), which carries binding sites for both activating and repressive transcription factors. We further hypothesize that repressive cellular transcription factors bind to the MIEP to mediate heterochromatinization of the genome in HPCs, and that HCMV reactivation requires 1) a switch in factors binding to the MIEP, from repressive to activating transcription factors; 2) recruitment of co-activator complexes through interaction with DNA-binding partners; 3) reprogramming of viral chromatin by enzymes that mediate histone modifications. We will investigate these hypotheses using experimentally infected CD34+ cells as a model for latency and differentiation to a dendritic cell phenotype as a model for reactivation. In Aim 1 we will 1) analyze modification of histones bound to viral genomes in latently infected hematopoietic progenitor CD34+ cells and in reactivated CD34-derived DCs; 2) correlate differentiation- dependent changes in the epigenome with changes in gene expression; and 3) investigate the requirement for H3K9 de-methylation in lytic infection of permissive cells and in reactivation in CD34-derived DCs. In Aim 2 we will investigate the role of transcription factors that bind to the MIEP in latency and reactivation. De- repression of early gene expression is a second key step in both lytic infection and in reactivation from latency. In Aim 3 we use a mutant virus that conditionally expresses IE proteins to investigate the role of the IE proteins in reprogramming viral chromatin to activate lytic replication. Through the use of state-of-the art epigenetics, proteomics, and functional analyses, our studies will have determined mechanisms that regulate viral chromatin in latency and reactivation, and identified potential new targets for therapeutic intervention to prevent reactivation of CMV. This project synergizes with Projects 1 and 3, which will investigate the roles of inflammation and epigenetics in control of MCMV in latency and reactivation.